The frontiers of molecular biology have necessitated high-throughput analytical technologies, capable of simultaneous identification and quantification of large numbers of biomolecule species. This covers the broad bioinformatics area of genomics, proteomics, intercellular and molecular signalling, metabolomics, cytomics and personalized medicine. For example, due to large variations of individuals' genetic signatures and frequent failures of traditional symptom-diagnostics, the emerging field of personalized medicine seeks to detect personal gene expression profiles, in order to focus upon therapies that are specific for each individual. Wider applicability of such personalized biomolecular diagnostics is currently hindered by issues such as assay speed and cost, as well as the complexity of the genome/proteome. An ideal tool would be a library or database of analytical channels, capable of supporting the analysis of not tens, but thousands of distinctive molecular targets.
In data storage, the main goal of multiplexing is to increase the data storage capacity within spatially-limited memory elements. In security printing of banknotes, identity cards, trademark tags, etc., multiplexing helps to prevents forgery, tampering or counterfeiting, and thermochromatic, magnetic, multi-colour fluorescent, and optically variable colour-changing inks have been used for this purpose. Multiplexing typically requires a matrix of optical codes, ideally carried by nano-/micro-sized objects, each of which should be accurately identifiable at high-speed and preferably in a low-cost fashion.
In principle, planar array biochips, used in multiplex assay techniques, provide infinite multiplexing capacity based on predetermined positions of microspots in the planar array. However in practice, this technology fails to provide sufficiently accurate quantitative data. For example, due to its requirement for high-precision robot encoding, diffusion concerns and/or varied biological environments between each reaction microspot. Moreover, relatively high manufacturing cost and a requirement that array plates be manufactured in fixed formats prior to analysis, interferes with or inhibits useful customisation.
Suspension arrays are emerging as a future leading technology for use in multiplex assay based molecular detection. Suspension arrays are based on ensembles of microspheres that are specially coded, most frequently by varying combinations of fluorescent dyes. The microspheres are thus endowed with a range of individually identifiable colour codes individually assigned to a specific analyte. Major advantages of suspension arrays include rapid reaction kinetics, the absence of washing leading to higher sample throughput, as well as reproducible manufacture of microsphere families. Suspension arrays are also providing potential for quantitative assays due to the uniform surface of microcarriers, simplicity of use and decreased expense compared to alternatives.
The level of spectral multiplexing available using suspension arrays is currently limited to around 100, achieved by encoding the microspheres with fluorescent dyes at varying intensity ratios. Producing a larger number of codes by using colours and intensities is difficult, due to the broad spectral width of fluorescent dyes and background fluorescence in the most popular microsphere material, polystyrene. Alternative methods include using differently sized and shaped microspheres, patterned reflective metal nanorods, micromachined signatures, spatially selective photobleached microspheres, rare-earth mass cytometry, and rare-earth doped glass microbarcodes. These approaches are considered unlikely to become mainstream due to various deficiencies (e.g. large size and/or high density materials).
Graphical encoding, for example designing particles as 1D barcodes, 2D or even 3D patterns, offers multiplexing capacity competitive to planar arrays. But the deciphering of graphic codes requires an active orientation mechanism and high-resolution pattern recognition, fundamentally restricting the analytical throughput. Electronic encoding can also generate numerous codes, but a primary weakness stems from a requirement for large dimensions (>100 μm). Other techniques, including spectrometric encoding making use of Raman scattering, IR absorption or mass spectroscopy fingerprints and physical encoding utilizing size, shape, and magnetism, are also of limited use due to respective limitations.
Optical encoding on the basis of digitized intensities at different fluorescence colour bands, is considered the most practical method. However, spectral overlapping of fluorescent probes becomes problematic when more than about ten intensities are desired, despite attempts to replace conventional organic dyes with novel materials such as quantum-dots. The multiplexing capacity of optical encoding remains practically limited to about 102. Decoding such a large number of codes presents another challenge. Although imaging-based decoding systems can, in principle, decode any suspension array, slight variations in the height of some particles on a planar surface will seriously interfere with an intensity-based decoding process.
Thus, there are outstanding problems to overcome or ameliorate so as to provide new or improved multiplex assays and/or arrays, for example to be able to increase the number of coding dimensions, in which the surface quantitative binding assays are immune to these codes. These codes should be able to be resolved under high-throughput conditions.
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